Hemifacial Spasm: gene expression and immunohistochemical analysis

Abstract

Purpose : No previous study assessed gene expression and immunohistochemical alterations in orbicularis oculi muscle (OOM) in affected and non-affected hemifacial spasm (HFS) patients, compared to samples obtained from healthy subjects. We aimed to investigate these differences.

Methods : High-throughput RNA sequencing was performed (Illumina Inc, San Diego, USA) in 18 orbicularis oculi samples from 6 untreated patients with HFS (both eyelids) and 6 controls (one eyelid). Over-Representation Analysis (ORA) using WebGestalt identified the most significant pathways and genes involved in HFS pathogenesis. Analogously, Immunohistochemistry of OOM from 6 HFS and 8 healthy subjects were prepared using monoclonal antibodies against Myosin Slow and Myosin Fast. (Novocastra, Leica, Newcastle, United Kingdom). The dilution of antibodies used was 1:100 (1 mL of antibody in 99 mL of diluent). An open digital image analysis software (ImageJ) was used for objective analysis.

Results : In the molecular aspect, out of 16,187 genes analyzed, 280 were found differentially expressed (DE) comparing the clinically affected side of HFS patients to controls’ eyelids. Threshold to log₂(fold-change) was 0.7 and p<0.05. DE upregulated genes were 239 and downregulated, 41. ORA revealed a profile in favor to upregulated muscle contraction, myogenesis and slow fiber transformation. In addition, OOM fiber diameter was significantly greater in both affected (p=0.003 and p=0.043 for fast and slow fibers, respectively) and non-affected sides (p=0.032 and p=0.044 for fast and slow fibers, respectively) of HFS samples. A significantly greater percentage of slow fibers was observed in the affected side of HFS patients (p=0.001) compared to control subjects.

Conclusions : This study's findings suggest that repeated chronic OOM contractions lead to fiber hypertrophy and possible conversion of fast-twitch into slow-twitch orbicularis oculi muscle fibers in patients with HFS. This hypothesis is corroborated not only in morphological, but also in molecular levels with gene expression alterations.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1. Summary of the RNA-seq results. Volcano plot representation of differential expression analysis of genes in the affected hemifacial spasm eyelid side versus controls. Red and blue points mark the genes with significantly increased or decreased expression respectively. The x-axis shows log₂(fold-changes) in expression and the y-axis the -log₁₀(p-values) are plotted.